Process for oligonucleo tide synthesis using phosphormidite intermediates

ABSTRACT

A new class of nucleoside phosphoramidites which are relatively stable to permit isolation thereof and storage at room temperature. The phosphoramidites are derivatives of saturated secondary amines.

The inventions described herein were made in the course of work under agrant or award from the Department of Health, Education and Welfare.

This is a divisional application of our earlier U.S. patent applicationSer. No. 637,927 filed on Aug. 6, 1984, now U.S. Pat. No. 4,668,777.U.S. patent application Ser. No. 637,927 is, in turn, a continuation ofU.S. patent application Ser. No. 358,589 filed on Mar. 16, 1982, nowabandoned. U.S. patent application Ser. No. 358,589 is, in turn, acontinuation-in-part of U.S. patent application Ser. No. 248,450 filedon Mar. 27, 1981, now U.S. Pat. No. 4,415,732.

This invention relates to new and useful Phosphorus compounds which areparticularly useful in the production of oligonucleotides.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new and useful phosphoramidites whichare intermediates for polynucleotide synthesis, as well as the improvedprocess for production of oligonucleotides from which polynucleotidesare prepared.

2. Description of the Prior Art

Numerous attempts have been made to develop a successful methodology forsynthesizing sequence defined oligonucleotides. However, the stepwisesynthesis of polynucleotides, and specifically oligonucleotides stillremains a difficult and time consuming task, often with low yields. Oneprior art technique has included the use of organic polymers as supportsduring polynucleotide synthesis. Classically the major problems withpolymer supported synthesis strategies has been inherent in the natureof the polymer support. Various prior art polymers used in suchsynthesis have proven inadequate for reasons such as: (1) slow diffusionrates of activated nucleotides into the support; (2) excessive swellingof various macroporous, sorption of reagent onto the polymer. See forexample, V. Amarnath and A. D. Broom, Chemical Reviews 77, 183-217(1977).

Modified inorganic polymers are known in the prior art, primarily foruse as absorption materials, for example, in liquid chromatography. Theattachment of nucleosidephosphates to silica gel using a trityl linkinggroup is described in the prior art (H. Koster, Tetrahedron Letters,1527-1530, 1972) but the method is apparently applicable only topyrimidine nucleosides. The cleavage of the nucleoside from the silicasupport can only be accomplished with acid to which the purinenucleosides are sensitive.

The production of phosphotriester derivatives of oligothymidylates isdescribed in literature (R. L. Letsinger and W. B. Lunsford, Journal ofthe American Chemical Society, 98:12, 3655-3661) by reaction of aphosphorodichloridite with a 5'-O blocked thymidine and subsequentreaction of the product with a 3'-O blocked thymidine followed byoxidation of the resulting phosphite to a phosphate and removal ofblocking groups to obtain the phosphotriesters; using this procedure,the tetramer and pentamer products, dTpTpTpT and dTpTpTpTpT in which Tis thymidine were prepared. Unfortunately, the process requiresseparation and purification of products at each stage to ensure propersequencing of the added nucleosides Separation techniques includingprecipitation and washing of precipitates are necessary to implementeach successive stage reaction.

In the aforementioned commonly assigned patent application are describedmethods for forming internucleotide bonds, i.e. bonds linkingnucleosides in an oligonucleotide or polynucleotide, by reaction ofhalophosphoridites with suitably blocked nucleoside or oligonucleotidemolecules.

The deoxynucleoside-modified silica gel is condensed with a selectednucleotide through formation of a triester phosphite linkage between the5' -OH of the deoxynucleoside. The phosphite linkage can be produced byfirst incorporating the phosphite group onto the 5' -OH of thenucleoside on the silica gel followed by condensation with the addednucleoside through the 3' -OH. Alternatively, and preferably, thephosphite group is incorporated into the added nucleoside at the ' -OH(the 5' -OH being blocked as by tritylating) and the resultingnucleoside phosphite then reacted with the 5' -OH of the nucleoside ofthe silica gel.

The deoxynucleoside-modified silica gel can also be condensed with aselected nucleoside through formation of a triester phosphite linkagebetween the 3' -OH of the deoxynucleoside of the silica gel and the 5'-OH of the selected deoxynucleoside. The phosphite linkage can beproduced by first incorporating the phosphite group onto the 3' -OH ofthe nucleoside on the silica gel followed by condensation with the addednucleoside through the 5' -OH. Alternatively and preferably by thisapproach, the phosp.hite group is incorporated into the added nucleosideat the 5' -OH (3' -OH being blocked as by tritylating using art formprocedures) and the resulting nucleoside phosphite then reacted with the3' -OH of the nucleoside on the silica gel.

The general reaction can be represented by the following: ##STR1##

The preferred reaction is represented as follows: ##STR2## wherein ○P isan inorganic polymer linked to the 3' or 5' -O- of the nucleosidethrough a base hydrolyzable covalent bond; R is H or a blocking group;R₁ ' is a hydrocarbyl radical containing up to 10 carbons; each B is anucleoside or deoxy-nucleoside base; and each A is H, OH or OR₄ in whichR₄ is a blocking group; and X is halogen, preferably Cl or Br or asecondary amino group.

The compounds of structure II and IIa wherein X is a 2° amino groupinclude those in which the amino group is an unsaturated nitrogenheterocycle such as tetrazole, indole, imidazole, benzimidazole andsimilar nitrogen heterocycles characterized by at least two ethylenicdouble bonds, normally conjugated, and which may also include otherheteroatoms such as N, S or O. These compounds of structure II and IIawherein X is such a heterocyclic amine, i.e., one in which the aminonitrogen is a ring heteroatom, are characterized by an extremely highreactivity, and consequently relatively low stability, particularly inthe indicated preparation of compounds of structure III and IIIa. Thesephosphoramidites and the corresponding chloridites from which they areprepared are unstable to water (hydrolysis) and air (oxidation). As aconsequence, such compounds can only be maintained under inertatmosphefe, usually in sealed containers, at extremely low temperaturesgenerally well below 0° C. Thus, the use of these compounds in thepreparation of compounds of structure III and IIIa requires extremeprecautions and careful handling due to the aforesaid high reactivityand low stability.

The present new compounds are of structure II and IIa wherein X is acertain type of secondary amino group. Specifically, the present newcompounds are those in which X is a saturated secondary amino group,i.e. one in which no double bond is present in the secondary aminoradical. More particularly, X is NR₂ 'R₃ ', wherein R₂ ' and R₃ ' takenseparately each represents alkyl, aralkyl, cycloalkyl andcycloalkylalkyl containing up to 10 carbon atoms, R₂ ' and R₃ ' whentaken together form an alkylene chain containing up to 5 carbon atoms inthe principal chain and a total of up to 10 carbon atoms with bothterminal valence bonds of said chain being attached to the nitrogen atomto which R₂ ' and R₃ ' are attached; and R₂ ' and R₃ ' when takentogether with the nitrogen atom to which they are attached form asaturated nitrogen heterocycle including at least one additionalheteroatom from the group consisting of nitrogen, oxygen and sulfur.

The present new compounds are not as reactive as those of the aforesaidcopending application and not as unstable. However, the present newcompounds do react readily with unblocked 3'-OH or 5'-OH of nucleosidesunder normal conditions. The present new phosphoramidites are stableunder normal laboratory conditions to hydrolysis and air oxidation, andare stored as dry, stable powders. Therefore, the present newphosphoramidites are more efficiently employed in the process of forminginternucleotide bonds, particularly in automated processing forformation of oligonucleotides and polynucleotides as described in theaforesaid copending application.

Amines from which the group NR₂ R₃ can be derived include a wide varietyof saturated secondary amines such as dimethylamine, diethylamine,diisopropylamine, dibutylamine, methylpropylamine, methylhexylamine,methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine,methycyclohexylmethylamine, butylcyclohexylamine, morpholine,thiomorpholine, pyrrolidine, piperidine, 2,6-dimethylpiperidine,piperazine and similar saturated monocyclic nitrogen heterocycles.

The nucleoside and deoxynucleoside bases represented by B in the aboveformulae are well-known and include purine derivatives, e.g. adenine,hypoxanthine and guanine, and pyrimidine derivatives, e.g. cytosine,uracil and thymine.

The blocking groups represented by R₄ in the above formulae includetrityl, methoxytrityl, dimethoxytrityl, dialkylphosphite, pivalyl,isobutyloxycarbonyl, t-butyl dimethylsilyl, acetyl and similar suchblocking groups.

The hydrocarbyl radicals represented by R₁ include a wide varietyincluding alkyl, alkenyl, aryl, aralkyl and cycloalkyl containing up toabout 10 carbon atoms. Representative radicals are methyl, butyl, hexyl,phenethyl, benzyl, cyclohexyl phenyl, naphthyl, allyl and cyclobutyl. Ofthese the preferred are lower alkyl, especially methyl and ethyl.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The preferred new compounds are those of structure IIa wherein X isdi-lower alkyl amino, pyrrolidino, morpholino or piperidino,particularly preferred being the lower alkyl amino especially,morpholino, dimethylamino and diethylamino; A is H; R₁ ' is lower alkyl;R is a trityl group; B is a nuceloside or deoxynucleotide base; and ○Pis silica gel.

The new compounds of the present invention can be prepared according toart-recognized procedures such as by reaction of the selected secondaryamine with the corresponding nucleoside phosphomonochloridite. Thisreaction is accomplished by dissolving the said nucleoside in an organicsolvent, such as tetrahydrofuran or acetonitrile, and adding theselected secondary amine. After removing unwanted hydrochloride salt,the organic solvent solution of the phosphoramidite may be used as suchfor polynucleotide synthesis or the product can be isolated from theorganic solvent solution and purified before further reaction.

As a further embodiment of the invention, the phosphoramidites arepreferably prepared by forming the desired chloro-(2°amino)alkoxyphosphine and thereafter condensing this product with theselected nucleoside. This procedure obviates the difficulties ofhandling inherent in the case of the nucleoside phosphomonochloroditewhich is susceptible to moisture hydrolysis and air degradation.

The reaction of the chloro-(2° amino)alkoxyphosphine is effected in anorganic solvent solution of the selected nucleoside, preferably in thepresence of a tertiary amine to take up the hydrogen chloride formed inthe condensation reaction. The reaction proceeds smoothly at roomtemperature in a dry atmosphere and under an inert gas such as N₂ orhelium. Organic solvents useful for this reaction include any solventwhich will dissolve the reactants such as diethyl ether, chloroform,methylene chloride, ethylene chloride, ethyl acetate, and the like. Thesolution of product is separated from the precipitated hydrochloridesalt of the added tertiary amine and can be used as such in formingpolynucleotide or alternatively can be separated from the solvent andpurified as by crystallization before further use. While the foregoingdisclosure has mentioned the use of chloro compounds, it should beunderstood that bromo compounds can be used as desired with essentiallythe same results.

When the present new compounds are used in forming internucleotidebonds, they are preferably employed with proton donors. Thus, thephosphoramidites are activated by acidic compounds through protonationwhich facilitates the desired internucleotide bond formation. The acidiccompounds to be employed for the purpose of the said activation arepreferably mildly acidic and include, for example, amine hydrohalidesalts and nitrogen heterocyclic compounds such as tetrazoles,imidazoles, nitroimidazoles, benzimidazoles and similar nitrogenheterocyclic proton donors. The amine hydrohalide salts to be used forthe protonation activation are preferably tertiary amine salts, and,preferably, the hydrochloride salts, although hydrobromide, hydroiodideor hydrofluoride salts can also be used. The aforesaid tertiary aminesinclude, for example, dimethylaniline, diisopropylaniline,methylethylaniline, methyldiphenylamine, pyridine and similar tertiaryamines.

When the nucleoside is guanosine, i.e. where B is guanine, the use ofamine hydrochlorides is not very effective for the purpose ofactivation, i.e. by protonation. With those compounds in which B isguanine, activation is preferably accomplished with the aforesaidnitrogen heterocyclic hydrogen donors.

Of course, as described in the aforesaid copending application, once theinternucleotide bond is formed, the product is then further treated toremove blocking groups, e.g. blocking group R, which permits reactionwith a further nucleoside of formula II herein and repeat reaction givesrise to the polynucleotide of determined sequence of nucleotidesattached to the silica gel through the covalently-bonded linking groups,e.g. ester linking group.

After each nucleoside is added, the phosphite group preferably should beoxidized to phosphate, usually by reaction with iodine as oxidizingagent, although this can be accomplished by reaction with peroxides suchas tertiary butyl peroxide and benzoyl peroxide, as well ashydroperoxides.

The oligonucleotide can then be obtained by hydrolytic cleavage toseparate from the silica gel support, usually after removal of blockinggroups such as R blocking groups and blocking groups on the nucleosidebase moieties as described in the aforesaid copending application,generally by hydrolysis. with ammonia.

Of particular value as blocking groups definitive of R are arylmethylgroups, including monoaryl dialkymethyl, diaryl monoalkylmethyl andtriarylmethyl blocking groups. Of these, the preferred are thetriarylmethyl of which the trityl blocking groups are well known. Thetriarylmethyl blocking groups are generally readily removable but theyalso afford a method of monitoring the sequencing of oligonucleotides aswell as the yield of product obtained. One major criticism of knownoligonucleotide synthesis is the lack of monitoring of the productproduced in successive condensations of nucleotides. Such monitoringwould require removal of an aliquot of the reaction system, e.g. thesilica gel or other support on which the oligonucleotide is beingsynthesized, hydrolysis of the product from the support and finallyanalysis of the product, all of which is time-consuming. Because of thisdifficulty, oligonucleotides are usually synthesized without appropriatemonitoring steps which is most undesirable. The use of triarylmethylblocking groups provides a simple but accurate method of monitoring thesequence of nucleosides in the oligonucleotide product as formed, aswell as the yield of product obtained at each stepwise addition ofnucleoside.

This method is predicated on color formation by the triarylmethyl cationin the presence of an acid, whether a Lewis acid or a protic acid. Byselection of appropriate triarylmethyl blocking groups for thephosphoramidite compound of structures II or IIa herein, which providedistinguishing color in acids, each nucleoside can be labelled with thetriarylmethyl group of distinguishing color. As each condensationreaction is completed to form the phosphorus linkage illustrated incompounds of formula III and IIIa herein, the next step in the synthesisis the removal of the blocking group R therefrom. This is convenientlyaccomplished with a Lewis acid such as zinc bromide and simultaneouslyproduces a color reaction, e.g. di-p-anisylphenylmethyl group forms anorange color with ZnBr₂, when removed from the oligonucleotide. Thecolor can be used to identify the triarylmethyl blocking group used toidentify the initial phosphoramidite employed and also to measure theextent of reaction by measurement of the intensity thereof.

Most triarylmethyl groups, in present experience, have shown colorproduction on exposure to acids. In fact, a wide variety of colors hasbeen obtained by varying the make-up of the triarylmethyl group,including as the aryl group not only phenyl and naphthyl but alsosubstituted phenyl and naphthyl, as well as heterocyclic rings such asquinolinyl, furyl, thienyl, and other nitrogen, sulfur and/or oxygencontaining heterocyclic rings. The said aryl groups can includesubstituents such as halide (F, Cl, Br, I); nitro, alkoxy, alkyl, aryl,aralkyl,cycloalkyl and like hydrocarbyl substituents. In thesesubstituents, the number of carbon atoms should preferably be from 1 toabout 12.

The preferred triarylmethyl groups are represented by the formula:##STR3## wherein each of R₁, R₂ and R₃ is an aryl group such as phenyl,naphthyl, quinolyl, furyl, thienyl, or other nitrogen, sulfur and/oroxygen-containing heterocyclic ring; or such aryl groups with amonosubstituent such as halide (F, Cl, Br or I), nitro, lower alkoxy,lower alkyl, and aryl, aralkyl and cycloalkyl containing up to 10 carbonatoms. R₂ and R₃ each may also be alkyl, cycloalkyl or aralkylcontaining up to 10 carbon atoms.

Preferable triarylmethyl groups are given in Table I:

                                      TABLE I                                     __________________________________________________________________________    LEGEND                                                                         ##STR4##                                                                      ##STR5##                                                                             ##STR6##                                                                                ##STR7##                                                                                 ##STR8##                                                                                ##STR9##                                                                                 ##STR10##                   (a)    (b)       (c)        (d)       (e)        (f)                           ##STR11##                                                                            ##STR12##                                                                               ##STR13##                                                                                ##STR14##                                                                               ##STR15##                                                                                ##STR16##                   (g)    (h)       (i)        (j)       (k)        (l)                           ##STR17##                                                                            ##STR18##                                                                               ##STR19##                                                                                ##STR20##                                                                               ##STR21##                                                                                ##STR22##                   (m)    (n)       (o)        (p)       (q)        (r)                          Aryl Functional Groups as Defined                                             in the Legend                   Color                                         __________________________________________________________________________    R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                                            Orange                                        R.sub.1 = c; R.sub.2 = b; R.sub.3 = a                                                                         Red                                           R.sub.1 = c; R.sub.2 = d; R.sub.3 = a                                                                         Orange                                        R.sub.1 = c; R.sub.2 = q; R.sub.3 = a                                                                         Colorless                                     R.sub.1 = c; R.sub.2 = r; R.sub.3 = a                                                                         Colorless                                     R.sub.1 = c; R.sub.2 = p; R.sub.3 = a                                                                         Red-Orange                                    R.sub.1 = R.sub.2 = b; R.sub.3 = a                                                                            Black                                         R.sub.1 = R.sub.2 = q; R.sub.3 = a                                                                            Colorless                                     R.sub.1 = R.sub.2 = r; R.sub.3 = a                                                                            Colorless                                     R.sub.1 = R.sub.2 = p; R.sub.3 =  a                                                                           Violet-Red                                    R.sub.1 = R.sub.2 = a; R.sub.3 = c                                                                            Yellow-Orange                                 R.sub.1 = R.sub.2 = a; R.sub.3 = b                                                                            Yellow                                        R.sub.1 = R.sub.2 = a; R.sub.3 = d                                                                            Yellow                                        R.sub.1 = R.sub.2 = a; R.sub.3 = q                                                                            Colorless                                     R.sub.1 = R.sub.2 = a; R.sub.3 = r                                                                            Colorless                                     R.sub.1 = R.sub.2 = c; R.sub.3 = n                                                                            Violet                                        R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                                            Blue                                          R.sub.1 = R.sub.2 = p; R.sub.3 = n                                                                            Deep Purple                                   R.sub.1 = R.sub.2 = c; R.sub.3 = o                                                                            Burnt Orange                                  R.sub.1 = R.sub.2 = c; R.sub.3 = p                                                                            Purple                                        R.sub.1 = R.sub.2 = b; R.sub.3 = p                                                                            Purple                                        R.sub.1 = R.sub.2 = g; R.sub.3 = m                                                                            Yellow-Orange                                 R.sub.1 = R.sub.2 = f; R.sub.3 = m                                                                            Colorless                                     R.sub.1 = R.sub.2 = p; R.sub.3 = m                                                                            Peach                                         R.sub.1 = R.sub.2 = e; R.sub.3 = m                                                                            Yellow                                        R.sub.1 = R.sub.2 = d; R.sub.3 = m                                                                            Yellow                                        R.sub.1 = R.sub.2 = c; R.sub.3 = m                                                                            Yellow                                        R.sub.1 = R.sub.2 = a; R.sub.3 = m                                                                            Colorless                                     R.sub.1 = R.sub.2 = b; R.sub.3 = m                                                                            Lilac                                         R.sub.1 = R.sub.2 = g; R.sub.3 = c                                                                            Red-Orange                                    R.sub.1 = R.sub.2 = f; R.sub.3 = c                                                                            Yellow                                        R.sub.1 = R.sub.2 = p; R.sub.3 = c                                                                            Red                                           R.sub.1 =  R.sub.2 = e; R.sub.3 = c                                                                           Red-Orange                                    R.sub.1 = R.sub.2 = d; R.sub.3 = c                                                                            Red                                           R.sub.1 = R.sub.2 = R.sub.3 = c Red                                           R.sub.1 = g; R.sub.2 = a; R.sub.3 = i                                                                         Deep Red                                      R.sub.1 = f; R.sub.2 = a; R.sub.3 = i                                                                         Yellow                                        R.sub.1 = p; R.sub.2 = a; R.sub.3 = i                                                                         Yellow                                        R.sub.1 = e; R.sub.2 = a; R.sub.3 = i                                                                         Red Violet                                    R.sub.1 = d; R.sub.2 = a; R.sub.3 = i                                                                         Burnt-Orange                                  R.sub.1 = c; R.sub.2 = a; R.sub.3 = i                                                                         Deep Purple                                   R.sub.1 = R.sub.2 = a; R.sub.3 = i                                                                            Red-Violet                                    R.sub.1 = b; R.sub.2 = a; R.sub.3 = i                                                                         Red                                           R.sub.1 = g; R.sub.2 = a; R.sub.3 = j                                                                         Yellow                                        R.sub.1 = f; R.sub.2 = a; R.sub.3 =  j                                                                        Yellow                                        R.sub.1 = p; R.sub.2 = a; R.sub.3 = j                                                                         Colorless                                     R.sub.1 = e; R.sub.2 = a; R.sub.3 = j                                                                         Orange                                        R.sub.1 = d; R.sub.2 = a; R.sub.3 = j                                                                         Carmine                                       R.sub.1 = c; R.sub.2 = a; R.sub.3 = j                                                                         Deep Burnt Orange                             R.sub.1 = R.sub.2 = a; R.sub.3 = j                                                                            Yellow                                        R.sub.1 = R.sub.2 = g; R.sub.3 = k                                                                            Yellow                                        R.sub.1 = R.sub.2 = f; R.sub.3 = k                                                                            Yellow                                        R.sub.1 = R.sub.2 = p; R.sub.3 = k                                                                            Colorless                                     R.sub.1 = R.sub.2 = e; R.sub.3 = k                                                                            Yellow-Orange                                 R.sub.1 = R.sub.2 = d; R.sub.3 = k                                                                            Yellow                                        R.sub.1 = R.sub.2 = c; R.sub.3 = k                                                                            Orange                                        R.sub.1 = R.sub.2 = a; R.sub.3 = k                                                                            Yellow                                        R.sub.1 = g; R.sub.2 = R.sub.3 =  a                                                                           Yellow                                        R.sub.1 = f; R.sub.2 = R.sub.3 = a                                                                            Yellow                                        R.sub.1 = p; R.sub.2 = R.sub.3 = a                                                                            Yellow                                        R.sub.1 = e; R.sub.2 = R.sub.3 = a                                                                            Orange                                        R.sub.1 = R.sub.2 = R.sub.3 = a Yellow                                        R.sub.1 = n; R.sub.2 = l; R.sub.3 = a                                                                         Green                                         R.sub.1 = h; R.sub.2 = l; R.sub.3 = a                                                                         Canary Yellow                                 R.sub.1 = g; R.sub.2 = l; R.sub.3 = a                                                                         Yellow                                        R.sub.1 = c; R.sub.2 = l; R.sub.3 = a                                                                         Yellow Orange                                 R.sub.1 = n; R.sub.2 = g; R.sub.3 = a                                                                         Green                                         R.sub.1 = h; R.sub.2 = g; R.sub.3 = a                                                                         Canary Yellow                                 R.sub.1 = R.sub.2 = g; R.sub.3 = a                                                                            Yellow                                        R.sub.1 = c; R.sub.1 = g; R.sub.3 = a                                                                         Yellow-Orange                                 R.sub.1 = b; R.sub.2 =  g; R.sub.3 = a                                                                        Yellow                                        R.sub.1 = n; R.sub.2 = R.sub.3 = g                                                                            Green                                         R.sub.1 = h; R.sub.2 = R.sub.3 = g                                                                            Canary Yellow                                 R.sub.1 = R.sub.2 = R.sub.3 = g Yellow                                        R.sub.1 = b; R.sub.2 = R.sub.3 = g                                                                            Yellow                                        R.sub.1 = n; R.sub.2 = j; R.sub.3 = a                                                                         Green                                         R.sub.1 = h; R.sub.2 = j; R.sub.3 = a                                                                         Canary Yellow                                 R.sub.1 = g; R.sub.2 = j; R.sub.3 = a                                                                         Yellow                                        R.sub.1 = c; R.sub.2 = j; R.sub.3 = a                                                                         Yellow-Orange                                 R.sub.1 = n; R.sub.2 = R.sub.3 = a                                                                            Green                                         R.sub.1 = h; R.sub.2 = R.sub.3 = a                                                                            Yellow                                        R.sub.1 = a; R.sub.2 = e; R.sub.3 = n                                                                         Green                                         R.sub.1 = a; R.sub.2 = e; R.sub.3 = h                                                                         Yellow                                        R.sub.1 = a; R.sub.2 = e; R.sub.3 = g                                                                         Yellow                                        R.sub.1 = a; R.sub.2 = e; R.sub.3 = c                                                                         Yellow-Orange                                 R.sub.1 = a; R.sub.2 = c; R.sub.3 = n                                                                         Red                                           __________________________________________________________________________

All colors were determined by the following procedure: an aliquot of thehydrolyzed Grignard reaction product (the triarylmethyl alcohol producedby the procedure described in Example V herein) was analyzed by thinlayer chromatography. The thin layer plates were then exposed tohydrochloric acid vapor and the color of the trityl cations recorded.

Thus, of the blocking groups definitive of R, the preferred are thearylmethyl groups, particularly triarylmethyl groups, and especiallythose arylmethyl groups which provide a visible color when contactedwith acids.

As used herein the symbols for nucleotides and polynucleotides andpolydeoxynucleotides are according to the IUPAC-IUB Commissioner ofBiochemical Nomenclature Recommendations [(1970) Biochemistry 9, 4022].

The following examples further illustrate the invention.

EXAMPLE 1

Preparation of phosphoramidites of the formula: ##STR23## represented ascompounds I-IV, in which in compound I, B=1-Thyminyl;

II, B=1-(N-4-benzoylcytosinyl);

III, B=9-(N-6-benzoyladeninyl);

IV, B=9-(N-2-isobutyrylguaninyl);

and DMT=di-p-anisylphenylmethyl.

The synthesis of compounds I-IV begins with the preparation of chloro-N,N-dimethylaminomethoxyphosphine [CH₃ OP(Cl)N(CH₃)₂ ] which is used as amonofunctional phosphitylating agent. A 250 ml addition funnel wascharged with 100 ml of precooled anhydrous ether (-78° C.) and precooled(-78° C.) anhydrous dimethylamine (45.9 g, 1.02 mol). The additionfunnel was wrapped with aluminum foil containing dry ice in order toavoid evaporation of dimethylamine. This solution was added dropwise at-15° C. (ice-acetone bath) over 2 h to a mechanically stirred solutionof methoxydichlorophosphine (47.7 ml, 67.32 g, 0.51 mol) in 300 ml ofanhydrous ether. The addition funnel was removed and the 1 l.,three-necked round bottom flask was stoppered with serum caps tightenedwith copper wire. The suspension was mechanically stirred for 2 h atroom temperature, then filtered and the amine hydrochloride salt washedwith 500 ml anhydrous ether. The combined filtrate and washings weredistilled at atmospheric pressure and the residue distilled underreduced pressure. The product was distilled at 40°-42° C. 13 mm Hg andwas isolated in 71% yield (51.1 g, 0.36 mol). d²⁵ =1.115 g/ml. ³¹P-N.M.R., =-179.5 ppm (CDCl₃) with respect to internal 5% v/v aqueous H₃PO₄ standard. ¹ H-N.M.R. doublet at 3.8 and 3.6 ppm J_(P-H) =14 Hz (3H,OCH₃) and two singlets at 2.8 and 2.6 ppm (6H, N(CH₃)₂). The massspectrum showed a parent peak at m/e=141.

The 4'-O-di-p-anisylphenylmethyl nucleoside (1 mmol) was dissolved in 3ml of dry, acid free chloroform and diisopropylethylamine (4 mmol) in a10 ml reaction vessel preflushed with dry nitrogen. [CH₃ OP(Cl)N(CH₃)₂ ](2 mmol) was added dropwise (30-60 sec) by syringe to the solution undernitrogen at room temperature. After 15 min the solution was transferredwith 35 ml of ethyl acetate into a 125 ml separatory funnel. Thesolution was extracted four times with an aqueous, saturated solution ofNaCl (80 ml). The organic phase was dried over anhydrous Na₂ SO₄ andevaporated to a foam under reduced pressure. The foam was dissolved withtoluene (10 ml) (IV was dissolved with 10 ml of ethyl acetate) and thesolution was added dropwise to 50 ml of cold hexanes (-78° C.) withvigorus stirring. The cold suspension was filtered and the white powderwas washed with 75 ml of cold hexanes (-78 ° C.). The white powder wasdried under reduced pressure and stored under nitrogen. Isolated yieldsof compounds I-IV were 90-94%. (see Table II). The purity of theproducts was checked by ³¹ P-N.M.R. Additionally, when analyzed by ³¹P-N M.R., these compounds were stable for at least a month when storedat room temperature under nitrogen. Furthermore, no significant amountof (3'-3')dinucleoside phosphite was detected by ³¹ P-N.M.R. (less than4%). The low content of the (3'-3') dinucleoside phosphite represents asignificant improvement over the prior art phosphite coupling procedurewhere a considerable amount of unwanted (3'-3') dinucleoside phosphitewas unavoidable.

The aminophosphoramidites I-IV were employed in condensation with3'-O-blocked nucleosides to form internucleotide bonds. Thephosphoramidites were activated by weak acids such as aminehydrochloride salts or tetrazoles.

A. In the following procedure, the process was monitored using ³¹ P-NM.R. In a 10 mm. N.M.R. tube, 1.2 molar equivalents of3'-O-levulinylthymidine and collidine were added to a mixture formed byadding N,N-dimethylaniline hydrochloride (1 mmol) in 0.5 ml dry CDCl₃ atroom temperature under N₂ to amidite compound I (0.5 mmol, -147.7 and-146.8 ppm) in 2 ml of dry, acid free CDCl₃ and an essentiallyquantitative yield of dinucleoside phosphite Ia (-140.8 and -139.9 ppm)was obtained.

B. Amidite compound I (0.5 mmol) and 3'-O-levulinylthymidine (0.6 mmol)were placed in a 10 mm N.M.R. tube and sublimed 1H-tetrazole (1.5 mmol)in 2.5 ml of dry acetonitrile-d₃ was added under nitrogen atmosphere.The ³¹ P-N.M.R. spectrum was immediately recorded and displayed aquantitative yield of Ia. Similarly, dinucleosides were obtained whenII, III and IV were reacted with 3'-levulinylthymidine to form IIa, IIIaand IVa as shown in Table II. The appropriate chemical shifts ofcompounds I-IV and Ia-IVa with respect to internal 5% v/v aqueous H₃ PO₄standard are reported in Table I.

                  TABLE II                                                        ______________________________________                                                                          ISOLATED                                              δ-.sup.31 P (ppm)                                                                   δ-.sup.31 P (ppm)                                                                   YIELD                                       COMPOUND  (Acetone-d.sub.6)                                                                         (CDCl.sub.3)                                                                              (%)                                         ______________________________________                                        I             -146.0, -145.4                                                                            -147.7, -146.8                                                                          93, 95*                                   II            -146.3, -145.5                                                                            -148.0, -147.0                                                                          92, 95*                                   III           -146.1, -145.8                                                                            -147.4, -147.3                                                                          90, 98*                                   IV            -145.9, -145.7                                                                            -147.7, -147.2                                                                          90, 98*                                   Ia            -139.6, -138.9                                                                            -140.8, -139.9                                                                          97**                                      IIa           -139.6, -139.0                                                                            -140.6, -140.0                                                                          94**                                      IIIa          -139.7, -138.9                                                                            -141.0, -139.9                                                                          97**                                      IVa           -140.3, -140.2                                                                            -143.6, -141.9                                                                          93**                                      ______________________________________                                         *Estimated purity from .sup.31 PN.M.R.                                        **Estimated yield from .sup.31 PN.M.R.                                   

EXAMPLE II Alternate procedure for Chloro-N,N-disubstitutedAminomethoxyphosphine

A 50 ml dropping funnel was charged with 31.59 g of N,N-Dimethylaminotrimethylsilane (42.1 ml, 0.27 mol) which wad addeddropwise over 1 h under nitrogen atmosphere to 25 ml of cold (-15° C.)methoxydichlorophosphine (35.15 g, 0.27 mol) in a 250 ml round bottomflask. A white unidentified precipitate formed during the course of theaddition. Once the addition was finished, the ice-acetone bath wasremoved and the suspension was stirred at room temperature for 1 h. Thereaction mixture was then slowly vacuum distilled through a one footlong, vacuum jacketed glass helices (3/32") column. The productdistilled at 40°-42° C. 13 mm Hg and was isolated in 81% yield (30.77 g,0.22 mol). d²⁵ =1.115 g/ml. ³¹ P-N.M.R., =-179.5 ppm (CDCl₃) withrespect to internal 5% aqueous H₃ PO₄ standard. ¹ H-N.M.R. doublet at3.8 and 3.6 ppm J_(P-4) =14 Hz (3H, OCH ) and two singlets at 2.8 and2.6 ppm (6H, N(CH₃) The mass spectrum showed a parent peak at m/e=141.Anal. calcd. for C₃ H₉ ClNOP: C, 24.45; H, 6.42; N, 9.90; O, 11.30; P,21.88. Found C, 24.53; H, 6.20; N, 10.04; O, 11.08; P, 21.44.

The procedure was successfully applied to the preparation of chloro-N,N-diethylaminomethoxyphosphine and chloropyrrolidino-methoxyphosphine.

EXAMPLE III

The applicability of phosphoramidites I-IV to the synthesis ofdeoxyoligonucleotides on polymer supports was accomplished by condensingcompounds I-IV with N-2-isobutyryldeoxyguanosine attached covalently tosilica gel. Thus, N-2isobutyryldeoxyguanosine (1 μmole) covalentlyattached to silica gel (20 mg) at the 3'-position, I (10 μmole), and1H-tetrazole (50 μmole in 0.1 ml dry acetonitrile) were shaken for minand the reaction was then quenched with aqueous lutidine. The samereaction sequence was effected with II, III and IV. After the usualoxidation and deprotection procedures, d(TpG), d(CpG), d(ApG) and d(GpG)were obtained in 100%, 98%, 94%, and yield respectively (measuredspectrometrically from the dimethoxytrityl cation using an extinction of7×10⁴ at 498 nm) These dinucleotides were completely degraded by snakevenom phosphodiesterase and the appropriate nucleosides and nucleotidewere obtained in the proper ratios (monitored via high pressure liquidchromatography analysis of snake venom phosphodiesterase hydrolysates).

The following deoxynucleotides have been synthesized using thisprocedure:

    __________________________________________________________________________    d(C--T--C--A--A--A--T--G--G--G--T--C)                                                                d(C--C--A--C--A--A--A--C--C--C)                        d(A--A--A--T--G--C--G--A--C--C--C--A)                                                                d(A--G--C--T--A--T--G--G--G--T--T--T)                  d(T--T--T--G--A--G--C--C--A--A--C--A)                                                                d(T--T--A--G--C--T--C--A--C--T--C--A)                  d(T--C--A--T--C--C--T--G--T--T--G--G)                                                                d(T--T--A--G--G--C--A--C--C--C)                        d(G--G--G--C--C--G--A--A--T--T--G--T)                                                                d(C--A--G--G--C--T--T--T--A--C--A)                     d(C--G--G--C--C--C--C--T--T--A--C--T)                                                                d(C--T--T--T--A--T--G--C--T--T--C)                     d(T--C--C--T--C--A--A--G--T--A--A--G)                                                                d(C--G--G--C--T--C--G--T--A)                           d(T--G--A--G--G--A--T--A--A--A--T--T)                                                                d(T--G--T--A--C--T--A--A--G)                           d(A--T--G--T--G--T--G--A--T--T--T--A)                                                                d(G--A--G--G-- T--T--G--T--A--T--G)                    d(G--T--G--G--T--A--A--A--T--C--A)                                                                   d(T--A--C--A--T--G--C--A--A)                           __________________________________________________________________________

EXAMPLE IV

5'-O-DMT-N-benzoyldeoxyadenosine [DMTrd(bzA)] (0.66 g., 1 mmole) in dryTHF (3 ml) is added dropwise under an argon atmosphere to a stirredsolution of the THF (3 ml) containing methyldichlorophosphite (0.113 ml,1.2 mmole) and 2, 4, 6 trimethylpyridine (0.633 ml. 4.8 mmole) at -78°C. After 10 minutes at -78° C., the reaction solution is filteredthrough a sintered glass funnel and solvent is removed by concentrationin vacuo. Excess methyl phosphodichloridite is removed by dissolving theresulting gum in toluene: THF (2 ml, 2:1) and re-evaporating in vacuo toa gum. This procedure is repeated several times to insured removal ofthe dichloridite. The nucleoside phosphomonochloridite is converted tothe tetrazolide. The gum resulting from the final re-evaporation isdissolved in THF (2 ml). A solution of the selected secondary amine 0.9mmole) in THF (2 ml) is then added dropwise with stirring at -78° C. tothe nucleoside phosphomonochloridite. After 10 minutes at -78° C., thesolution is transferred to a centrifuge tube, spun at low speed, and thesupernatant is removed. This solution contains the activated nucleosidephosphoramidite. If not used immediately, this phosphoramidite can beplaced in long term storage after precipitation by dropwise additioninto dry pentane, followed by collection, drying in vacuo and storing insealed tubes under argon or other inert gas at room temperature, orlower temperatures, e.g. 0° C. All operations are performed under inertgas to avoid oxidation. At no time is the active agent exposed to air.

The foregoing procedure is applicable for the preparation of activatedthymidine, deoxycytidine, and deoxyadenosine nucleotides. For thepreparation of the activated deoxyguanosine nucleotide, the procedure isthe same except for the stoichiometry. The molar ratio of5'-O-DMT-N-isobutyryldeoxyguanosine [DMTrd(ibG)];methyldichlorophosphite; 2, 4, 6 trimethylpyridine and tetrazole is1:0.9:3.8:0.7. The steps necessary for addition of one nucleotide to themodified silica gel polymer support follow. The removal of thedimethoxytrityl group from the nucleotide is accomplished by exposingthe modified silica gel support to 0.1M ZnBr₂ in nitromethane for 15 to30 minutes The support is then washed initially with butanol: 2, 6lutidine:THF (4:1:5 by volume) and finally with THF. The solvent ratiois not important since this step is used to remove potential zinc estersof nucleosides. This step- could be eliminated but lower yields mayresult. Other Lewis acids could be substituted for ZnBr₂, such as BF₃,AlCl₃ and TiCl₄. However ZnBr₂ is preferred. Protic acids can also beused. However approximately 3-5% depurination of each purine by proticacids is observed even whe the amount of acid is reduced to the minimumamount needed to remove the dimethoxytrityl group. The next step in theprocess is condensation of the protected and activated nucleotide to thenucleoside or oligonucleotide covalently bound to the support. This isaccomplished by using 10-15 equivalents of the activated phosphoramiditeand a reaction time of about one hour. The solvent is anhydrous THF. Thenext step in the process is the blocking of unreacted 5'-hydroxylgroups. This is accomplished using a solution of acetic anhydride,dimethylaminopyridine, pyridine and THF. This may also be accomplishedusing a 0.33M solution of diethylmonotriazolophosphite in2,6-lutidine/THF (1:5 by volume). The reaction time is 5 min. and isfollowed by a THF wash. As a further alternative, a solution ofphenylisocyanate/lutidine (45:55 by volume) and a 90 minute reactiontime may be used for this step. This solution is then removed from themodified silica gel by washing the support with THF and withacetonitrile. The first procedure is preferred. This step can beeliminated or other reagents that react with 5'-hydroxyl groups and arecompatible with the overall chemistry can be substituted therefore.However, by including this step, the final purification of the desirableoligonucleotide is rendered much easier. This is because the complexityof the total synthetic material bound to the support is reducedconsiderably. The final step in each cycle is oxidation of the phosphiteto the phosphate. A composition of 0.1M I₂ in water/2, 6 lutidine/THF(1:1:3) is preferred, although other ratios can be used. Furthermore,other oxidizing agents such as N-chlorosuccinimide or aryl or alkylperoxides, e.g., t-butyl peroxide, could also be used. After theaddition of the appropriate activated nucleotides in any predeterminedsequence, the deoxyoligonucleotide is removed from the support by basehydrolysis and blocking groups where present are also removed, eitherselectively i.e., stepwise, or in an overall hydrolysis treatment suchas heating at 50° C. in ammonium hydroxide. When R₁ is a methyl group,this is removed by treatment with thiophenol prior to removing theoligonucleotide from the support.

EXAMPLE V General method for synthesizing chlorotriarylmethanes

In the synthesis of this series of compounds there are two types ofsubstrates for the respective Grignard reagents: (1) diaryl ketones,i.e. benzophenones, which require one equivalent of Grignard reagent;(2) esters of aryl carboxylic acids, which require two equivalents. Thefollowing will describe the former. Appropriate adjustments should bemade for reactions of the latter type.

                  TABLE VII                                                       ______________________________________                                        A Summary of Reagents Used for Synthesizing                                   Triarylcarbinols                                                              Reagent  Example         mmoles                                               ______________________________________                                        aryl bromide                                                                           p-bromoanisole  100                                                  magnesium                110                                                  diethyl ether                      250 ml                                     iodine                             2 crystals                                 diaryl ketone                                                                          4-methoxybenzophenone                                                ______________________________________                                    

The magnesium, aryl bromide and ether are combined in 1000 ml roundbottom flask. The iodine is added. In order to initiate the formation ofthe aryl magnesium bromide, it is necessary to crush the magnesium witha glass rod. [Note: grinding the magnesium in a Waring Blender alsohelps to get the reaction going.] Once the reaction has begun, it isallowed to reflux, with no external heating, until the ether ceases toboil. An ethereal solution of the diarylketone is added dropwise, withstirring. The reaction is allowed to proceed overnight. At this time thereaction is analyzed by thin layer chromotography (tlc) in 1:1ether:hexane The R_(f) of the product will be approximately 0.7.

If the reaction is satisfactory, it is quenched with 10% (w/v) ammoniumsulfate. The product is extracted four times with 300 ml of toluene. Theextracts are dried over sodium sulfate and evaporated down as far aspossible. The concentrated organic phase is dried in vacuo overnight. Atthis time the product crystallizes out. The product tritanol iscollected in a funnel and washed with hexane.

The tritanol is taken up in 100 ml of toluene. 200 mmoles of acetylchloride is added. 300 ml of hexane is added. The product is allowed torecrystallize overnight at -20° C. The crystals are collected, washedwith hexane, and dried in vacuo.

In order to determine the reactivity of the trityl chloride, a smallamount is quenched into water and N-butanol with toluene as solvent. Thesamples are analyzed via tlc using 3:1 hexane:ether. The trityl butylether runs at R_(f) approximately 0.8 while the tritanol runs at R_(f)approximately 0.4.

Using this procedure, the various alcohols described in Table I wereprepared.

Several of the triarylmethylchlorides were condensed with the 5'hydroxyl of appropriately protected deoxynucleosides. These compoundsare listed in Tables IV and V. The 5'-triarylmethyldeoxynucleosides weretreated with protic and Lewis acids using carefully controlledconditions. The results of these studies are also recorded in Tables IVand V. These results show that several triarylmethyl groups formingdifferent colors in acid solutions are hydrolyzed at approximately thesame rapid rate in the presence of ZnBr₂. The rates are more variable-with protic acids.

                  TABLE IV                                                        ______________________________________                                        The Lewis Acid Hydrolysis Rates of Triarylmethyl Groups                       Attached to the 5'-Hydroxyl of Deoxynucleosides.sup.1                                                     t1/2 (sec)                                                                             Color                                    Triarylmethyl Group.sup.2                                                                    Deoxynucleoside.sup.3                                                                      in ZnBr.sub.2                                                                          in Acid                                  ______________________________________                                        R.sub.1 = n; R.sub.2 = c; R.sub.3 = a                                                        T            60       Green                                    R.sub.1 = n; R.sub.2 = e; R.sub.3 = a                                                        T            60       Red                                      R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           T            60       Orange                                   R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                           T            30       Blue                                     R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           C            45       Orange                                   R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                           C            30       Blue                                     R.sub.1 = R.sub.2 = b; R.sub.3 = a                                                           G            20       Black                                    R.sub.1 =  R.sub.2 = c; R.sub.3 = a                                                          G            20       Orange                                   R.sub.1 = h; R.sub.2 = R.sub.3 = a                                                           A            45       Yellow                                   R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           A            20       Orange                                   ______________________________________                                         .sup.1 Reaction conditions were 0.08 M ZnBr.sub.2 in nitromethane.            Aliquots were removed from the reaction solution, quenched with ammonium      acetate and analyzed visually after tlc and exposure to HCl vapors. Time      points were taken at 10, 20, 30, 45, 60, 90, 120, 180, and 240 sec.           .sup.2 The aromatic functional groups are defined in the legend to Table      1.                                                                            .sup.3 The symbols T, C, G, and A refer to the nucleosides thymidine,         Nbenzoyldeoxycytidine, Nisobutyrldeoxyguanosine, and                          Nbenzoyldeoxyadenosine. The nucleoside 5hydroxyl was derivatized to           contain the triarylmethyl group.                                         

For the repetitive addition of mononucleotides to a growingoligonucleotide attached covalently to a polymer support. the variouscolor coded triarylmethyl groups should preferably be hydrolyzed atapproximately the same rate. Otherwise, each addition cycle must beindividually monitored if completed manually or independently programmedif completed in a machine. Because the hydrolysis rates with ZnBr aresimilar, the results outlined in Table IV suggest that most, if not all,of the triarylmethyl alcohols listed in Table 1 could be incorporatedinto synthetic procedures as color coded blocking groups.

                  TABLE V                                                         ______________________________________                                        The Protic Acid Hydrolysis Rates of Triarylmethyl Groups                      Attached to the 5'-Hydroxyl of Deoxynucleosides.sup.1                                                              Time                                                    Deoxy-          Color (sec) to                                                nucleo- t1/2 (sec)                                                                            in    Complete                                 Triarylmethyl Group.sup.2                                                                    side.sup.3                                                                            in H+   Acid  Hydrolysis                               ______________________________________                                        R.sub.1 = n; R.sub.2 = c; R.sub.3 = a                                                        T       30      Green 45                                       R.sub.1 = n; R.sub.2 = e; R.sub.3 = a                                                        T       180     Red   >600                                     R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           T                                                              0-             Orange  30                                                     R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                           T       45      Blue  90                                       R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           C                                                              0-             Orange  30                                                     R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                           C       45 to   Blue  120                                                             60                                                     R.sub.1 = R.sub.2 = b; R.sub.3 = a                                                           G       15      Black 45                                       R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           G                                                              0-             Orange  30                                                     R.sub.1 = h; R.sub.2 = R.sub.3 = a                                                           A       60      Yellow                                                                              240                                      R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           A                                                              0-             Orange  30                                                     ______________________________________                                         .sup.1 Reaction conditions were 2% toluenesulfonic acid in                    chloroform:methanol (7:3). Aliquots were removed from the reaction            solution, quenched with ammonium acetate and analyzed visually after tlc      and exposure to HCl vapors. Time points were taken at 15, 30, 45, 60, 90,     120, 240, 300, and 600 sec.                                                   .sup.2 The aromatic functional groups are defined in the legend to Table      1.                                                                            .sup.3 The symbols T, C, G, and A refer to the nucleosides thymidine,         Nbenzoyldeoxycytidine, Nisobutyrldeoxyguanosine, and                          Nbenzoyldeoxyadenosine. The nucleoside 5hydroxyl was derivatized to           contain the triarylmethyl group.                                         

                  TABLE VI                                                        ______________________________________                                        Table VI provides the spectral characteristics of                             selected triarylmethyl alcohols.                                                                         Extinction                                                        λ Maximum(s).sup.2                                                                 Coefficient                                        Triarylcarbinol.sup.1                                                                        (nanometers)                                                                              (Molar.sup.-1 cm.sup.-1)                           ______________________________________                                        R.sub.1 = R.sub.2 = b; R.sub.3 = a                                                           423          9300                                                             503          5200                                                             586          3900                                              R.sub.1 = R.sub.2 = a; R.sub.3 = h                                                           452         42000                                              R.sub.1 = a; R.sub.2 = c; R.sub.3 = n                                                        545         25000                                                             455         28000                                              R.sub.1 = R.sub.2 = b; R.sub.3 = N                                                           586         15500                                              R.sub.1 = a; R.sub.2 = n; R.sub.3 = e                                                        577          9500                                                             421         20500                                              ______________________________________                                         .sup.1 See the legend to Table 1 for a definition of the functional group     R.sub.1, R.sub.2 and R.sub.3.                                                 .sup.2 All spectra were taken in a saturated ZnBr.sub.2 nitromethane          solution. All spectra were recorded on a Carey model 21, scanning from 35     nm to 600 nm.                                                            

Four deoxyoligonucleodides were synthesized using color codeddeoxynucleotide phosphoramidites. The compound wered(G-T-A-T-A-A-C-A-C), d(C-A-T A-A A-G-A-A-A-A-A),d(G-T-A-C-A-G-C-T-G-G-C-T) and d(C-C-C-T-T-T-C-T-T-A-A-A). The5'-hydroxyl of each deoxynucleotide was protected with a differenttriarylmethyl group. These groups as assigned for the synthesis ofdeoxyoligonucleotides are listed in Table VII.

                  TABLE VII                                                       ______________________________________                                        Triarylmethyl Group.sup.1                                                                    Deoxynucleoside  Color.sup.2                                   ______________________________________                                        R.sub.1 = R.sub.2 = b; R.sub.3 = n                                                           N-benzoyldeoxycytidine                                                                         Blue                                          R.sub.1 = h; R.sub.2 = R.sub.3 = a                                                           N-benzoyldeoxyadenosine                                                                        Yellow                                        R.sub.1 = c; R.sub.2 = n; R.sub.3 = a                                                        Deoxythymidine   Red                                           R.sub.1 = R.sub.2 = c; R.sub.3 = a                                                           N-isobutyrldeoxyguanosine                                                                      Orange                                        ______________________________________                                         .sup.1 The aromatic functional groups are defined in the legend to Table      I.                                                                            .sup.2 The color of the triarylmethyl group is observed when the              5triarylphenyl deoxynucleoside is exposed to either protic or Lewis acids                                                                              

Thus the 5'-triarylmethyl groups of N-benzoyldeoxyadenosine,N-benzoyldeoxycytidine, N-isobutyrldeoxyguanosine and deoxythymidineproduced yellow, blue, orange and red colors respectively when exposedto either Lewis or protic acids. These triarylmethyldeoxynucleosideswere synthesized as outlined in this disclosure. Conversion to theappropriate 5'-O-triarylmethyl and deoxynucleoside N,N-dimethylaminomethoxyphosphines was completed using the procedure ofExample VI.

EXAMPLE VI General synthesis of 5'-triarylmethyl deoxynucleosides

5 mmoles of N-protected deoxynucleoside or thymidine is dissolved in 50ml of dry pyridine. The sample is evaporated to a gum in vacuo. 25 ml ofdry pyridine is added. Six mmoles of triarylmethyl chloride is added.The reaction mixture is shaken overnight. The reaction is monitored inmethanol:chloroform (1:9). The product has an R_(f) of 0.5 and theunreacted deoxynucleoside has an R_(f) of 0.2. The reaction is quenchedwith 5 ml of absolute methanol.

After 30 minutes the reaction mixture is concentrated to a small volume,taken up in ethyl acetate and extracted once with water. The organicphase is dried over sodium sulfate and concentrated to a gum. 10 ml oftoluene is added and then evaporated.

The reaction mixture is then taken up in chloroform and applied to asilica gel column (5 cm×20 cm) that has been equilibrated with 1%pyridine in chloroform. After the compound is loaded on the column, thecolumn is washed with 500 ml of 1% pyridine in chloroform. The compoundis eluted from the column with 3 to 6% methanol. The fractionscontaining the desired product are pooled, concentrated to a foam, takenup in chloroform and precipitated into hexane.

The precipitate is collected in a Buchner funnel and dried in vacuo. Theaverage yield by weight is 85%.

The 5'-triarylmethyldeoxynucleosides carrying functional groups asoutlined in Table VII were connected tochloro-N,N-dimethylaminomethoxyphosphine using the procedure of ExampleI. The 5'-triarylmethyldeoxynucleoside-3'-N,N-dimethylaminomethoxyphosphines were used as intermediates indeoxyoligonucleotide synthesis using the procedure of Example IV. Thus,the synthesis of d(G-T-A-T-A-A-C-T-A-C-A-C) begins withN-benzoyldeoxycytidine attached covalently to silica gel through the3'-hydroxyl. The next step was condensation with5'-O-p-tolyldiphenylmethyl-N- benzoyl-deoxyadenosine 3'-N,N-dimethylaminomethoxyphosphine. After acylation and oxidation,detritylation was completed using a saturated solution of ZnBr₂ innitromethane:methanol (19:1). A yellow color indicating the addition ofN-benzoyldeoxyadenosine was observed. The remaining nucleotides wereadded in a similar manner. During each detritylation step, colors wereobserved in the following sequential order: blue, yellow, red, blue,yellow, yellow, red, yellow, red, and orange. These were the expectedcolors and confirm that the correct deoxyoligonucleotide wassynthesized. Purification of the deoxyoligonucleotide was by reversephase high performance liquid chromatography and polyacrylamide gelelectrophoresis. Characterization was by two dimension sequence analysis(Sanger, Donelson, Coulson, Kossel, and Fischer, Proc. Natl. Acad. Sci.USA 70, 1209-1213, 1973). This analysis reconfirmed that the correctdeoxyoligonucleotide had been synthesized as indicated by thecolorimetric results. The three remaining deoxyoligonucleotides weresynthesized and characterized in the same way.

For the synthesis of the four enumerated oligodeoxynucleotides, thequantities of silica gel used and the choice of nucleoside joined to thesilica gel support are summarized in Table VIII.

                                      TABLE VIII                                  __________________________________________________________________________                           Nucleoside on Silica                                                                       μmole Nucleoside/                                                                    Gram Silica                     Deoxyoligonucleotide   Gel          Gram Silica Gel                                                                         Gel Used                        __________________________________________________________________________    d(G--T--A--T--A--A--C--T--A--C--A--C)                                                                N-benzoyldeoxycytidine                                                                     45        0.15                            d(C--A--T--A--A--A--G--A--A--A--A--A)                                                                N-benzoyldeoxyadenosine                                                                    40        0.15                            d(C--C--C--T--T--T--C--T--T--A--A--A)                                                                N-benzoyldeoxyadenosine                                                                    40        0.15                            d(G--T--A--C--A--G--C--T--G--G--C--T)                                                                deoxythymidine                                                                             53        0.15                            __________________________________________________________________________

Table IX summarizes physical parameters of5'-O-triaylmethylnucleoside-3,-N,N-dimethylaminomethoxyphosphines usedin the synthesis of the four enumerated oligodeoxynucleotides.

                                      TABLE IX                                    __________________________________________________________________________                           Phosphorus NMR Chemical                                Nucleotide         M. Wt.                                                                            Shifts (ppm).sup.1                                                                          Color.sup.2                              __________________________________________________________________________    5'-O-di-ρ-anisylphenylmethyl-                                                                746 146.3, 146.1  Orange                                   N-isobutyryldeoxyguanosine-3'-N,                                              N-dimethylaminomethoxyphosphine                                               5'-O-ρ-anisyl-1-naphthylphenyl-                                                              659 146.4, 145.7  Red                                      methyldeoxythymidine-3'-N, N-                                                 dimethylaminomethoxyphosphine                                                 5'-O-di-ο-anisyl-1-napthylmethyl-                                                        790 147.6, 145.4  Blue                                     N-benzoyldeoxycytidine-3'-N, N-                                               dimethylaminomethoxyphosphine                                                 5'-O-ρ-tolyldiphenylmethyl-N-benzoyl-                                                        718 146.4, 146.1  Yellow                                   deoxyadenosine-3'-N, N-dimethylamino-                                         methoxyphosphine                                                              __________________________________________________________________________     .sup.1 Spectra were recorded in CH.sub.3 CN as solvent and against            phosphoric acid as external standard.                                         .sup.2 Color produced in either a Lewis acid or a protic acid.           

For each condensation step, 120 μmoles of the5'-O-triarylmethylnucleotide, acetonitrile, and 480 μmole tetrazole wereused. The next steps were acylation with acetic anhydride, oxidationwith I₂ and detritylation with ZnBr₂. After each detritylation step, theexpected color corresponding to the required trityl cation was observed.

Once each synthesis was complete, the deoxyoligonucleotide was isolatedby the following procedure. Each deoxyoligonucleotide covalently boundto silica gel (30 mg) was first treated withthiophenol:triethylamine:dioxane (1:1:2) for 90 minutes, washed fourtimes with methanol and then washed once with diethylether. The silicagel was isolated by centrifugation and air dried. Each sample was nexttreated with t-butylamine:methanol (1:1) for 18 hours at 50° C. Thesupernatants obtained after centrifugation were removed and dried invacuo. The silica gel samples were next treated with concentratedammonium hydroxide at room temperature for three hours in order toremove the deoxyoligonucleotide from the silica gel. The supernatantswere transferred to test tubes containing the residues from thet-butylamine procedure and the solutions concentrated in vacuo. Freshconcentrated ammonium hydroxide was added to the dry residues and thesolutions were warmed at 50° C. for 22 hours in order to remove aminoprotecting groups from deoxyoligonucleotide bases. The samples wereconcentrated in vacuo and each sample was next dissolved in 200 μlwater. Purification was by reverse phase high performance liquidchromatography. The retention times and solvent conditions are outlinedin Table X. Each deoxyoligonucleotide was next treated with 80% aceticacid at room temperature for 1 hour in order to remove thetriarylphenylmethyl group. After concentration in vacuo, each sample waspurified by polyacrylamide gel electrophoresis and analyzed as to thecorrect deoxymononucleotide sequence by two dimension sequence analysis.

                  TABLE X                                                         ______________________________________                                                                  %                                                                             Ace-   Reten-                                                                 toni-  tion                                         Deoxyoligonucleotide      trile.sup.1                                                                          Time.sup.2                                   ______________________________________                                        d(G--T--A--T--A--A--C--T--A--C--A--C).sup.3                                                             29     2.6                                                                    27     3.8                                                                    26     6.2                                          d(C--A--T--A--A--A--G--A--A--A--A--A).sup.4                                                             30     2.9                                                                    28     3.0                                                                    26     4.5                                          d(C--C--C--T--T--T--C--T--T--A--A--A).sup.4                                                             30     2.9                                                                    26     4.6                                                                    25     7.2                                                                    24     9.8                                          d(G--T--A--C--A--G--C--T--G--G--C--T).sup.5                                                             29     2.6                                                                    27     3.6                                                                    25     6.3                                          ______________________________________                                         .sup.1 The aqueous buffer contains 0.1 M triethylammonium acetate.            .sup.2 1.2 min/k' at 2.0 ml/min.                                              .sup.3 Triarylmethyl group was dianisylphenylmethyl, preparative isolatio     was at 25% acetonitrile                                                       .sup.4 Triarylmethyl group was dianisyl-1-napthylmethyl, preparative          isolation was at 25% acetonitrile.                                            .sup.5 Triarylmethyl group was dianisylphenylmethyl, preparative isolatio     was at 24% acetonitrile.                                                 

What is claimed is:
 1. In a process for producing oligonucleotides whichcomprises the step of condensing the 3'-OH or 5'-OH group of anucleoside or oligonucleotide with a nucleoside phosphite, theimprovement being that the nucleoside phosphite is a compound of one ofthe formulae: ##STR24## wherein B is a nucleoside or deoxynucleosidebase; A is H OH or OR₄ in which R₄ is a blocking group; R is a blockinggroup; R₁ ' is a hydrocarbyl radical containing up to about carbonatoms; and X is NR₂ 'R₃ ', wherein R₂ ' and R₃ ' taken separately eachrepresent alkyl, aryl, aralkyl, cycloalkyl and cycloalkylalkylcontaining up to 10 carbon atoms; R₂ ' and R₃ ' when taken together forman alkylene chain containing up to 5 carbon atoms in the principal chainand a total of up to 10 carbon atoms with both terminal valence bonds ofsaid chain being attached to the nitrogen atom to which R₂ ' and R₃ 'are attached; and R₂ ' and R₃ ' when taken together with the nitrogenatom to which they are attached form a saturated nitrogen heterocycleincluding at least one additional heteroatom from the group consistingof nitrogen, oxygen and sulfur.
 2. The process according to claim 1wherein R is a trityl group.
 3. The process according to claim 1 whereinR is a di-p-anisylphenylmethyl.
 4. The process according to claim 1wherein R is p-anisyldiphenylmethyl.
 5. The process according to claim 1wherein R₁ ' is lower alkyl.
 6. The process according to claim 1 whereinX is di-lower alkylamino.
 7. The process according to claim 1 wherein Xis a saturated nitrogen heterocyclic.
 8. The process according to claim1 wherein B is adenine, guanine, cytosine, uracil or thymine.
 9. Theprocess according to claim 1 wherein R is di-p-anisylphenylmethyl, B is9-(N-6-benzoyladeninyl), R₁ ' is methyl, A is H and X is morpholino. 10.The process according to claim 1 wherein R is di-p-anisylphenylmethyl, Bis thyminyl, R₁ is methyl, A is H and X is dimethylamino.
 11. Theprocess according to claim 1 wherein R is di-p-anisylphenylmethyl, B is1-(N-4-benzoylcytosinyl), R₁ ' is methyl, A is H and X is dimethylamino.12. The process according to claim 1 wherein R isdi-p-anisylphenylmethyl, B is 9-(N-6-benzoyladeninyl), R₁ ' is methyl, Ais H and X is dimethylamino.
 13. The process according to claim 1wherein R is di-p-anisylphenylmethyl, B is 9-(N-2-isobutyrylguaninyl),R₁ ' is methyl, A is H and X is dimethylamino.
 14. The process accordingto claim 1 wherein said nucleoside or oligonucleotide is covalentlybonded to an inorganic polymer.
 15. The process according to claim 14wherein said nucleoside or oligonucleotide is linked to the inorganicpolymer through a base hydrolyzable covalent bond.
 16. The processaccording to claim 15 wherein the base hydrolyzable covalent bond is anester linkage.